Homocysteine assay

ABSTRACT

The present invention provides an improved method of assessing/quantifying the amount of homocysteine in a body fluid sample via an enzymatic assay which comprises reducing background signal by treatment with one of the following: a reducing agent, a pyruvate deactivating agent, heat treatment, or by lyophilising or immobilizing the homocysteine converting enzyme.

[0001] The present invention relates to improvements in and relating toenzymatic assays for homocysteine in biological fluid samples.

[0002] Elevated blood plasma homocysteine levels can be correlated torisk of cardiovascular disease, e.g. coronary heart disease, coronaryartery disease, cerebrovascular disease, and peripheral vasculardisorders. Indeed elevated homocysteine levels are thought to be abetter predictor of cardiovascular disease than elevated cholesterollevels. Generally plasma levels of 15 μM and below are consideredhealthy (see for example New England Journal of Medicine 1997, 337:230).

[0003] Accordingly there is a need for reliable methods for determininghomocysteine levels in patients.

[0004] Direct determination of homocysteine has thus far proved to becomplicated, e.g. because it has not been found possible to raise anantibody against homocysteine (HCy) which is not cross-reactive withother substances present in biological samples.

[0005] However, in WO 93/15520 (Axis) and WO 98/07872 (Glasgow) forexample, homocysteine assays are described which involve enzymaticconversion of homocysteine and determination of homocysteine levels bydetermination of a homocysteine conversion product produced by theenzyme-mediated conversion.

[0006] Such assays require the use of a reducing agent (e.g.dithiothreitol) to liberate covalently bound homocysteine and of ahomocysteine converting enzyme and while they perform well there is roomfor improvement in terms of increasing signal to noise, e.g. by reducingbackground signal.

[0007] We have now surprisingly found that the performance of suchenzymatic HCy assays may be improved by a variety of relatively simplemeasures.

[0008] A first of such measures involves treatment of the biologicalfluid sample with a reducing agent for example a thiol (particularly adithiol, such as dithiothreitol (DTT), dithioerythrol (DTE) orbis-(2-mercaptoethyl)sulphone), phosphine (e.g.triscarboxyethylphosphine (TCEP) or tri-n-butyl-phosphine), methyliodide, thioredoxin, lipoic acid or a borohydride) to liberatecovalently bound homocysteine, addition of a homocysteine convertingenzyme and then treatment of the sample with an agent which neutralizesthe reducing agent, e.g. one which binds to it, oxidizes it or otherwisedepotentiates it, for example an organic disulphide compound or adithiol (especially DTT or DTE) binding agent.

[0009] Viewed from one aspect therefore the invention provides an assayfor homocysteine which comprises contacting a biological fluid samplewith a reducing agent, especially DTT or DTE, and subsequently withhomocysteine desulphurase, characterised in that said sample iscontacted with an agent which binds, oxidizes or depotentiates saidreducing agent, e.g. an organic disulphide or a dithiol (especially DTTor DTE) binding or depotentiating agent, for example cystamine or amaleimide, after being contacted with said homocysteine desulphurase.

[0010] Examples of suitable binding agents for thiol-based reducingagents such as DTT and DTE include maleimides, particular cyclicN-maleimides, i.e. optionally 3 and or 4-substituted, N-substituted1-aza-2,5-dioxo-cyclopentenes, particularly compounds in which theN-substitution is to produce a bis maleimide and wherein the ring C andN substituents contain up to 25 carbons (e.g. comprising alkyl, aryl,aralkyl, aralkyl and maleimide groups). Examples of particularmaleimides include N-methyl-maleimide, N-ethyl-maleimide,1,1′-(3,3′-dimethyl-1,1′-biphenyl-4,4′-diyl)-bismaleimide,1,1′-(methylene di-4,1-phenylene)bismaleimide,N-(1-phenylethyl)maleimide, 1-(2-methoxy-5-methyl phenyl) maleimide, and2-methyl-N-phenyl-maleimide. Many of these compounds are availablecommercially, e.g. from Sigma Aldrich. Examples of other suitabledithiol depotentiating agents include cystamine and other compounds thatreduce the reducing capacity of dithiols such as DTT, oxiranes,aziridines, aryl halides, mercurials (e.g. p-chloromercuribenzenesulphonic acid and hydroxymercuribenzoic acid), vinyl sulphones,haloacetyl compounds (e.g. iodoacetimide), 5,5-dithio-bis(2-nitrobenzoic acid) (i.e. Ellman's Reagent) and disulphide exchange reagentssuch as pyridyl sulphides (e.g. 4,4-dipyridyl-disulphide).

[0011] The reducing agent (e.g. DTE, DTT or TCEP) is conveniently addedto the biological fluid sample at a concentration of 0.5 to 5 mM,especially preferably about 1 mM. Where the reducing agent is not adithiol, an organic disulphide rather than a dithiol binding ordepotentiating agent will be used. The organic disulphide or dithiolbinding or depotentiating agent is typically added to a concentration of0.05 to 20 times the reducing agent concentration used, preferably 0.1to 10 times, particularly 0.2 to 1.0 times, especially 0.3 to 0.6 times.Thus it will generally be added to a concentration of 0.05 to 200 mM,especially 0.2 to 15 mM, particularly 0.2 to 1.0 mM for dithiol bindingagents (e.g. maleimides) and 0.2 to 200 mM, especially 1 to 100 mM,particularly 5 to 15 mM for organic disulphides (e.g. cystamine).

[0012] The biological fluid sample used in the assay of the invention isconveniently a blood or blood derived sample, e.g. plasma or serum,although other biological fluids may be used if desired. The sample ispreferably cell-free, e.g. being prepared by centrifugation, filtration,or by lysis.

[0013] The assays of the invention preferably involve the use of asecond enzyme for which a product of the enzyme-mediated homocysteineconversion is a substrate. If necessary further enzymes or other systemsmay be used to generate a directly detectable analyte. Especiallypreferably the second enzyme is lactate dehydrogenase (LDH), which inthe presence of nicotinamide adenine dinucleotide (NADH) convertsα-ketobutyrate to α-oxo-butyrate and NAD⁺. NAD⁺ can be detected by acolour generating cycling reaction as described in WO 98/07872 (Glasgow)and in the Examples below.

[0014] The neutralization or depotentiation of DTT in such HCy assayssignificantly improves the assay result by reducing background; howeverthere is still room for further improvement.

[0015] The homocysteine desulphurase enzyme used in such HCy assays isgenerally unstable on storage in aqueous solution and thus are generallyprovided in lyophilized form, e.g. using albumin, for example bovineserum albumin (BSA) as the cryo/lyoprotectant. We have now surprisinglyfound that assay background may be reduced by avoiding use of BSA, e.g.by use instead of thiol-free cryo/lyoprotectants, e.g. thiol-freealbumin, immunoglobulins, polyalkyleneoxides (e.g. PEG), or sugars suchas trehalose and maltose.

[0016] Thus viewed from a further aspect the invention provides ahomocysteine assay which comprises contacting a biological fluid samplewith a liquid reagent containing a homocysteine converting enzyme,especially HDS, wherein said reagent is produced by adding an aqueousliquid to a lyophilisate containing said enzyme and acryo/lyoprotectant, characterised in that said lyophilisate issubstantially free of thiol-containing cryo/lyoprotectants.

[0017] In this aspect of the invention, the enzymes used may be enzymesas described in WO 93/15220 (Axis), WO 98/07872 (Glasgow), U.S. Pat. No.5,985,540 (Anti Cancer), U.S. Pat. No. 5,885,767 (Biocatalytics), U.S.Pat. No. 5,998,191 (Anti Cancer), and the publications referred totherein.

[0018] The homocysteine converting enzyme is preferably one whichremoves or converts the thiol group in HCy. Examples of homocysteineconverting enzymes include S-adenosyl homocysteine hydrolase (SAHH),homocysteinase or homocysteine desulphurase (HDS),dimethylthetin-homocysteine methyl transferase (DHMT), methioninesynthetase (MS) and cystathionine β-synthetase (CβS). Preferably howeverthe enzyme is homocysteine desulphurase (HDS), which convertshomocysteine to α-ketobutyrate.

[0019] Examples of suitable cryo/lyoprotectants (otherwise referred toas bulking agents or stabilizers) include thiol-free albumin,immunoglobulins, polyalkyleneoxides (e.g. PEG), trehalose, mannitol,glucose, maltose, raffinose and stachyose. (See for example WO97/29782). These can be used in conventional amounts in conventionallyophilization techniques.

[0020] This second aspect of the invention is especially preferably usedin combination with the first aspect of the invention.

[0021] Where second or further enzymes are used in lyophilised form,these too are preferably prepared using thiol-free cryo/lyoprotectants.

[0022] Alternatively the HDS containing reagent may be provided as aliquid containing a thiol-reducing agent (e.g. DTT, DTE, TCEP, etc.),together with a proteinaceous or non-proteinaceous stabilizer, e.g.thiol-free albumin (e.g. DTT or TCEP treated albumin) or immunoglobulin(e.g. IgG, for example bovine gamma globulin), peptones or apolyalkylene oxide (such as a PEG) or a polyol such as a fatty acidester (e.g. C₁₆₋₂₂, especially C₁₈ fatty acid) of a polyol (especially aC₆ polyol) or a polyoxyethylated derivative thereof (e.g. a Span orTween nonionic surfactant such as Tween 20), a carbohydrate orpolysaccharide. In general, proteinaceous stabilizers should be used atconcentrations of up to 10% wt, e.g. 0.01 to 10%, preferably 0.01 to 1%,more preferably 0.02 to 0.5%, e.g. 0.05 to 2 mg/mL.

[0023] Thus viewed from a further aspect the invention provides ahomocysteine assay which comprises contacting a biological fluid samplewith a liquid reagent containing homocysteine desulphurase, wherein saidliquid reagent is an aqueous liquid containing homocysteinedesulphurase, a thiol-reducing reagent (e.g. at 0.05 to 20 mM,especially 0.05 to 15 mM, preferably 1 to 10 mM), and a proteinaceous ornon-proteinaceous stabilizer (e.g. at 0.01 to 10% wt, preferably 0.01 to1%, more preferably 0.02 to 0.5%).

[0024] The three aspects of the invention described above serve toreduce the background signal level, i.e. the signal generated byperformance of the assay in the absence of the homocysteine conversionenzyme. However again there is still room for further improvement.

[0025] It has surprisingly been found that background levels may bereduced still further by treatment of the biological fluid sample toremove pyruvates and other keto acids.

[0026] Thus viewed from a further aspect the invention provides ahomocysteine assay which comprises contacting a biological fluid samplewith a homocysteine converting enzyme, characterised in that beforecontact with said enzyme said sample is treated with an agent whichserves to deactivate pyruvates, e.g. by immobilizing, binding orconverting pyruvates.

[0027] Examples of agents which deactivate pyruvates includenon-enzymatic agents such as thiamine, alkaline hydrogen peroxide,dichloromethyl ether, air or oxygen, or nucleophiles such as hydrazine,semicarbazide and hydroxylamines and enzymes such as pyruvatecarboxylase, pyruvate oxidase, malate dehydrogenase, transaminases,acetoacetate decarboxylase, lactate dehydrogenase, pyruvatedecarboxylase, 2-ketobutyrate dehydrogenase, alanine transaminase,acetolactate synthase, 2-ethylmalate synthase, urocanate hydralase,cystathione lyase, methylalanine dehydrogenase,N5-(carboxyethyl)-ornithine synthase, methylmalonyl Co-A carboxytransferase, glutamine-pyruvate transaminase, pyridoxamine-pyruvatetransaminase, serine-pyruvate transaminase, lysine-pyruvatetransaminase, ATP:pyruvate 2-O-phosphotransferase, and especiallyalanine amino transferase and pyruvate dehydrogenase. Thiamine promotesnon-enzymatic decarboxylation of pyruvates. Hydrazines serve to trappyruvates. Pyruvate decarboxylase and the coenzyme thiaminepyrophosphate convert pyruvate to acetaldehyde and CO₂. Alanine aminotransferase, together with glutamate and pyridoxyl-5-phosphate, convertspyruvates to 2-oxo-glutarate and L-alanine, and pyruvate dehydrogenaseconverts pyruvates to acetyl Co-A.

[0028] If the agent used to deactivate pyruvate is hydrogen peroxide, itwill be understood that the hydrogen peroxide will need to beneutralised prior to contacting the sample with said enzyme. Anysuitable reagent i.e. antioxidant or enzyme may be used, but preferablythe hydrogen peroxide is neutralised using catalase, which catalyzes thefollowing reaction: 2H₂O₂→2H₂O+O₂.

[0029] Preferably, hydrogen peroxide is added to the sample at 0.01% to1% final concentration, preferably 0.05% to 0.5% and most preferably 0.1to 0.3%. Catalase is added after the removal of pyruvate at 10 to 500U/ml, preferably 50 to 400 U/ml, most preferably 80 to 300 U/ml.

[0030] Some pyruvate-converting enzymes such as malate dehydrogenase andpyruvate dehydrogenase are NAD dependent for their action and thus maybe less preferred where the homocysteine assay involves a NAD dependantsignal generation step.

[0031] Enzymatic pyruvate-converting agents will generally be preferredover non-enzymatic agents due to their relative specificity as a resultof which any excess agent will not give rise to undesired effects laterin the assay procedure. Thus for example where hydrazine is used and thehomocysteine assay involves use of HDS to generate a-ketobutyrate, thea-ketobutyrate must be protected by removal of any remaining hydrazinesor other nucleophiles, e.g. by addition of ketones or more preferablyaldehydes (e.g. acetone or formaldehyde) in stoichiometric amounts afterthe pyruvate-conversion step.

[0032] Typically, the biological fluid sample will be contacted with:0.1 to 20 mM hydrazine (especially 0.1 to 15 mM, particularly 0.5 to 10mM); 20 to 150 μM glutamate (especially 25 to 100 μM, particularly 30 to80 μM), 1 to 100 μM pyridoxyl-5-phosphate (especially 5 to 80 μM,particularly 10 to 50 μM) and 5 to 50 IU alanine amino transferase; 1 to100 mM pyruvate dehydrogenase (especially 1 to 60 mM, particularly 5 to50 mM) and 1 to 100 μM Co Enzyme A (especially 5 to 80 μM, particularly10 to 50 μM); or 1 to 100 mM pyruvate decarboxylase (especially 1 to 60mM, particularly 5 to 50 mM) and 1 to 100 μM thiamine pyrophosphate(especially 5 to 80 μM, particularly 10 to 50 μM). Appropriateconcentrations for such pyruvate-deactivating agents can be determinedby routine experimentation on pyruvate-containing biological fluidsamples.

[0033] Pyruvate removal may be effected using several reagents andseveral treatment steps if desired. Thus such treatment may for exampleinvolve:

[0034] i) pre-treatment of patient samples with acid (e.g. 1M HCl)followed by centrifugation and subsequent use in the assay of thesupernatant. The acid will typically be added to achieve a concentrationin the supernatant of 0.1 to 1M, preferably 0.2 to 0.5M, especially 0.2to 0.3M;

[0035] ii) heat-treatment, e.g. to 37 to 100° C., preferably 37 to 80°C., more preferably 40 to 60° C., of the plasma or serum;

[0036] iii) esterification of pyruvic acid, e.g. by addition of ethanoland HCl;

[0037] iv) removal of carboxylic acid groups, e.g by addition of 0.1 to3 mg/mL EDAC, preferably 0.1 to 2 mg/mL, especially 0.5 to 1.5 mg/mL,and Tris buffer, e.g. 0.1 mM Tris, pH 7;

[0038] v) addition of semicarbazide, e.g. to a concentration of 0.01 to10 mM, preferably 0.1 to 5 mM, especially 0.2 to 2 mM;

[0039] vi) addition of hydroxylamine, e.g. to a concentration of 0.01 to10 mM, preferably 0.1 to 5 mM, especially 0.2 to 2 mM.

[0040] vii) pretreatment with LDH (generally to a concentration of 10 to40 μg/mL, preferably 12 to 30 μg/mL, especially 15 to 25 μg/mL) and NADH(generally 20 to 80 μM, preferably 45 to 55 μM) to convert pyruvate tolactate, and heat treatment (e.g. as above preferably in the presence ofnitrous acid) to remove NAD+;

[0041] viii) pre-treatment with β-naphthylamine (generally to aconcentration of 0.1 to 10 mg/mL, preferably 0.1 to 5 mg/mL, especially0.2 to 1 mg/mL);

[0042] ix) if required treatment to remove NAD, e.g. by heating asdescribed above, heating in an acid environment, or exposure toultraviolet light; and

[0043] x) pre-treatment of patient samples via exclusion filtrationfollowed by centrifugation in order to remove pyruvate and other ketoacids. Any suitable filter may be used.

[0044] Pyruvate removal may be effected using size exclusion filters,together with centrifugation. Any suitable exclusion filter may be used,e.g. 10 kD to 60 kD exclusion filter, preferably 20 to 50 kD, mostpreferably 30 kD. Use of exclusion filtration followed by centrifugationprior to the addition of the homocysteine converting enzyme forms apreferred aspect of the invention.

[0045] Filtering the biological fluid sample through a molecular sievein order to remove or reduce background signals in the enzymatichomocysteine assay forms a preferred aspect of the invention.

[0046] Following the deactivation of pyruvate, it forms a preferredaspect of the invention to heat the sample, e.g. to 37 to 100° C.,preferably 37 to 80° C., more preferably 40 to 60° C., for 5 to 100minutes, preferably 10 to 80 minutes, more preferably 15 to 60 minutes.It forms a further preferred aspect if the pyruvate deactivating agentused prior to heat treatment is hydrogen peroxide.

[0047] This aspect of the assay of the invention may be performed usingany of the homocysteine converting enzymes mentioned in the publicationsreferred to above, especially HDS. This aspect of the invention ispreferably used together with the first, second or third aspects,especially with both the first and second or first and third aspects.

[0048] Preferably, a microtitre plate is used in any of the aspects ofthe invention.

[0049] Further, the use of immobilized homocysteine converting enzymesmay allow the removal of pyruvate from the serum.

[0050] Thus, viewed from a further aspect the invention provides ahomocysteine assay which comprises contacting a biological fluid samplewith an immobilized homocysteine converting enzyme, especially HDS,wherein said biological fluid sample contacts the immobilized enzymeunder such time and conditions to allow the homocysteine in the sampleto bind to said enzyme, characterised in that the biological fluidsample is then removed from the assay.

[0051] In this aspect of the invention, the enzymes used may be asdescribed previously. Preferably however the enzyme is HDS.

[0052] The homocysteine-converting enzyme may be immobilized by anysuitable technique well known in the art. Preferably said homocysteineconverting enzyme is attached to a solid support, via any suitablelinkage. As used herein “linkage” refers to any interaction between thehomocysteine converting enzyme and the solid support, enabling them tobe associated. Such interaction may involve physical association such ascovalent binding and may also involve so-called “weak” interactions suchas hydrogen bonds, Van der Waals forces and ionic interactions.Alternatively, the homocysteine converting enzymes may be provided withmeans for attachment to a solid support. Such means may constitute orcomprise, for example, one partner of an affinity binding pair, e.g.biotin, binding to the corresponding binding partner of the affinitybinding pair, i.e. streptavidin, provided on the solid support. DNA:DNAbinding proteins and antibodies:antigens may also be used as alternativebinding pairs.

[0053] Alternatively, the solid support may be provided with means forattachment to a homocysteine converting enzyme. Suitable means includeimmobilized antibodies or fragments thereof, amine binding ligands orprotein binding ligands e.g. plates coated with chelating nickel whichare commercially available e.g. plates coated with chelating nickelwhich are commercially available (Qiagen Ltd, UK). In order to create anamine binding surface the solid support may be succinimide activatedmicrotitre plates can either be coated with succinimide, or arecommercially available (Pierce Chemical Company, US).

[0054] The solid support may be any of the well known supports ormatrices which are currently widely used or proposed for immobilisation,separation, etc. These may take the form of particles, sheets,dipsticks, gels, filters, membranes, fibres, capillaries or microtitrestrips, tubes, plates or wells etc.

[0055] Conveniently, the support may comprise glass, silica, latex or apolymeric material as for example nitrocellulose, teflon, alginate,agarose, polystyrene, latex or nylon. Preferred are materials presentinga high surface area for binding.

[0056] Preferably, a microtitre plate may be used in the method of theinvention.

[0057] The attachment of the homocysteine converting enzyme to a solidsupport allows easy manipulation of the enzyme. Thus, the attachment tosome kind of solid support can enable the separation of the homocysteinefrom the rest of the components in the sample, including pyruvate. Thiscan be achieved for example by carrying out washing steps.

[0058] The attachment of the homocysteine converting enzyme to a solidsupport avoids the need to use the lyophilised form of the enzyme.

[0059] Preferably, the biological fluid sample is treated with areducing agent such as DTT, DTE or TCEP prior to contact with theimmobilized homocysteine converting enzyme, in order to releasecovalently bound homocysteine. The sample is then contacted with theimmobilized enzyme under such conditions to allow homocysteine tobinding to said enzyme, i.e. for 0.5 to 10 minutes, preferably 1 to 8minutes, more preferably 1 to 5 minutes. Once the homocysteine has boundto the homocysteine converting enzyme, the solid support may be washedin order to remove the biological fluid sample. The solid support iswashed using any suitable fluid, preferably a buffer solution, mostpreferably phosphate buffer.

[0060] After the biological fluid sample has been removed from theimmobilized enzyme, the assay may be as described previously.

[0061] Preferably, the immobilized enzyme is HDS. In order to facilitatethe method of this aspect of the invention, the active site or substratebinding site of the enzyme have been engineered by any suitable means,for example via genetic engineering of recombinant enzyme DNA, or bychemical treatment of the homocysteine converting enzyme, in order tochemically alter the active or binding site. With regard to HDS,treatment of the immobilized enzyme with hydrazine prior to contactingthe enzyme with the biological fluid sample, removes pyridoxal 5phosphate from the active site of the enzyme. Once the biological fluidsample has been removed from the immobilized homocysteine sample, thepyridoxal 5 phosphate moiety can be reintroduced. Removal of thepyridoxal 5 phosphate moiety from the active site of the HDS enzymeallows homocysteine to bind to HDS, but no reaction can take place untilthe pyridoxal-5-phosphate moiety is replaced. Therefore, the engineeringof the enzyme to allow binding of homocysteine, but preventing thefurther enzymatic reaction, is a preferred aspect of the invention. Oncethe pyruvate has been removed via washing, the enzyme is treated toenable the reaction to proceed.

[0062] This aspect of the invention is preferably used together with thefirst aspect of the invention.

[0063] Where the biological fluid on which an enzymatic homocysteineassay is carried out is serum or plasma, we have also found thatbackground, or more precisely patient-to-patient background variation,may be reduced if the cells are removed from the sample rapidly afterblood collection, preferably within 60 minutes, more preferably within30 minutes or less (i.e. 25, 20, 15, 10, 5 minutes or less). Accordinglyit is proposed that blood samples for enzymatic assay for homocysteineshould be filtered cell free, e.g. through a needle equipped with afilter, within 60 minutes of collection. This forms a further aspect ofthe present invention.

[0064] Viewed from this aspect the invention provides an enzymatic assayfor homocysteine in a blood sample, characterised in that said sample isfiltered cell-free (i.e. red and white blood cell free) within 60minutes, preferably within 30 minutes, of being withdrawn from thepatient.

[0065] Filtering according to this aspect of the invention may beeffected by passing the blood sample through a filter, for example afilter in a syringe needle base or in a sample receiving tube, or byabsorption in an absorbent web followed by compression of the wetted webto expel a cell-free fluid sample.

[0066] The background in a enzymatic homocysteine assay may bedetermined by performing the assay without use of the homocysteineconverting enzyme.

[0067] In the assays of the invention, the homocysteine concentrationmay be determined in a qualitative, semi-quantitative or quantitativemanner, e.g. as an absolute concentration or as an indication thatconcentration is above or below a threshold value or inside or outside aparticular range. Total homocysteine concentration will generally bedetermined; however if desired treatment with a reducing agent such asDTT, DTE or TCEP may be omitted and the free homocysteine concentrationmay instead be determined. To allow quantitative determination, theassay will preferably be calibrated by being run with standardscontaining homocysteine at a known concentration or more preferably aseries of known concentrations. The background signals for the varioussamples tested will likewise preferably be determined so that acorrected signal may be generated by subtraction of the backgroundsignal from the detected signal. Preferably sample signals, backgroundsignals and calibration signals will be detected following performanceof the assay for the same period of time, i.e. after allowing signal tobuild up for the same period of time. For each type of assay, theoptimum period of time for signal build up may be determined readily byfollowing the time-dependence of signal and background development. Suchan optimum may obviously be a balance between signal value and assayduration, especially where the assay is performed on automatedapparatus.

[0068] The signal determined in the assay will depend on the nature ofthe reaction used to generate the signal. Thus the signal will generallybe a radiation (e.g. light) absorption, emission or scattering. In theexamples below, the signal is the light absorption (generally measuredat 550 nm) by the colored formazan compound generated from a colourlesstetrazolium salt. In this particularly preferred form of the assay ofthe invention, the following process steps and reactions occur:

[0069] 1. A blood sample is collected and separated into plasma orserum.

[0070] 2. The cells from the sample are removed, preferably within 60minutes, more preferably within 30 minutes, e.g. by centrifugation orfiltration.

[0071] 3. The resultant plasma or serum sample is preferably treatedwith a pyruvate removing agent, e.g. hydrogen peroxide.

[0072] 4. Excess pyruvate removing agent is removed (i.e. excesshydrogen peroxide is removed with catalase).

[0073] 5. The sample is preferably treated with a reducing agent,especially preferably dithiothreitol (DTT), dithioerythrol (DTE), ortriscarboxyethylphosphine (TCEP) to release covalently boundhomocysteine.

[0074] 6. The sample is contacted with HDS to convert HCy toα-ketobutyrate, H₂S and ammonia.

[0075] 7. The HDS-treated sample is preferably treated with an organicdisulphide (especially where a non-sulphide reducing agent is used instep (4)), or an oxidizing agent (e.g. perchloric acid, iodate orperiodate), or more preferably a DTT binding agent (e.g. a maleimide),to bind or destroy excess reducing agent.

[0076] 8. The sample is contacted with NADH and lactate dehydrogenase(LDH) and incubated to convert α-ketobutyrate and NADH toα-hydroxybutyrate and NAD⁺.

[0077] 10. The sample is acidified to remove excess NADH and thenbrought to neutral pH.

[0078] 11. The sample is contacted with ethanol, a tetrazolium salt,alcohol dehydrogenase (ADH) and an oxidizing agent causing an NAD⁺/NADHcycling reaction where NAD⁺ and ethanol are converted to NADH andacetaldehyde and NADH and the tetrazolum salt are converted to NAD⁺ andformazan. The mixture is incubated and the formazan concentration ismeasured by light absorption at 550 nm, generally after addition of acidto destroy NADH and stop the cycling reaction.

[0079] In the scheme set out above, the sample is referred to as beingcontacted with the various reagents at the relevant stages of thereaction scheme. However in order to reduce the total number of reagentsolutions required, certain reagents may and generally will be added atearlier stages.

[0080] Viewed from a further aspect, the invention also provides a kitfor a homocysteine assay, said kit comprising:

[0081] homocysteine desulphurase, preferably (i) in lyophilized form,the lyophilisate being substantially free of thiol-containingcryo/lyoprotectants or (ii) in aqueous liquid form further containing adithiol reducing agent (e.g. DTT, DTE or TCEP) and a proteinaceous ornon-proteinaceous stabilizer;

[0082] a L-homocyst(e)ine (or L homocysteine precursor) standard,preferably a plurality of standards containing L-HCy or L-homocystine(or a precursor) at a plurality of known concentrations;

[0083] a reducing agent, e.g. dithiothreitol, dithioerythiol, TCEP ormethyl iodide;

[0084] an agent which binds, oxidizes or depotentiates the reducingagent, e.g. an organic disulphide or a dithiol binding agent, preferablya maleimide;

[0085] optionally one or more further reagents capable of converting thehomocysteine conversion product of HDS into a detectable analyte (e.g.LDH, NADH, ADH, a tetrazolum salt, an oxidizing agent, and an acid);

[0086] preferably a pyruvate deactivating agent, e.g. hydrazine,acetoacetate decarboxylase, pyruvate carboxylase, hydrogen peroxide orpyruvate dehydrogenase;

[0087] optionally one or more additional reagents capable ofremoving/inactivating the pyruvate deactivating agent, i.e. catalase;and

[0088] optionally a filter capable of removing red blood cells fromblood.

[0089] The publications mentioned herein are hereby incorporated byreference.

[0090] The invention will now be illustrated further by the followingnon-limiting Examples, with reference to the drawings in which:

[0091]FIG. 1 shows the comparison of background signal with and withoutmaleimide and BSA, as a plot of mean OD 550 nm versus time in minutes.Three plots are shown, demonstrating the data obtained from Example 5,(A) ♦ no maleimide plus BSA, (B) ▪ maleimide plus BSA and (C) ▴maleimide without BSA. The experimental conditions are defined inExample 5;

[0092]FIG. 2 shows the correlation of results fromhomocysteine-containing samples from Example 6 read on IMX and by theenzymatic method, as a plot of IMX value versus estimated concentrationvia enzymatic assay in μM. The experimental conditions are defined inExamples 6, 7 and 8.

[0093]FIG. 3 shows the results from Example 6, the effect of removal ofpyruvate with pyruvate carboxylase, as a plot of OD 550 nm versusconcentration of homocysteine in μm. Four plots are shown, ♦ sodiumbicarbonate, ATP and pyruvate carboxylase, ▪ sodium bicarbonate, ATPwithout pyruvate carboxylase, ▴ potassium bicarbonate, ATP and pyruvatecarboxylase, and X potassium bicarbonate, ATP without pyruvatecarboxylase.

EXAMPLE 1

[0094] Assay Reagents

[0095] A) Pyruvate and Keto Acid Remover

[0096] 0.47% Hydrogen peroxide

[0097] (Can use 0.47% to 10%) B) Enzyme Reagent 1 Homocysteinedesulphurase 0.02 U/mL Lactate dehydrogenase 20.8 μg/mL NADH   50 μMCryo/lyoprotectant*  0.8 wt % Phosphate buffer (pH 8.0)  0.1 M catalase 300 U/ml Total volume  1.5 mL

[0098] The reagent is in lyophilized form and may be reconstituted with1.5 mL Ro grade water. It is then stable for 8 hours.

[0099] C) Blank Reagent 1

[0100] As enzyme reagent 1 but without the homocysteine desulphurase.

[0101] D) Reducing Reagent

[0102] Dithiothreitol 20 mM in aqueous 2.5 mM citric acid, pH 3.0.

[0103] E) Reagent 2

[0104] 190 mM HCl containing 0.55% wt Nonidet P40, 1.5 mM maleimide, and5.5% ethanol

[0105] F) Reagent 3

[0106] 16 μM MPMS (1-methoxy-5-methyl-phenazinium methyl sulfate); 144μM NBT, 26U/ml ADH, in 963 mM Tris buffer pH 7.6.

[0107] 3.25 mL of 0.1 M phosphate buffer, pH 7.0 (0.026 g disodiumhydrogen orthophosphate and 0.016 g sodium dihydrogen orthophosphate1-hydrate, in Ro grade water), containing 1% sucrose (maltose ortrehalose) (32 mg), 23.7 U/ml ADH (alcohol dehydrogenase) and 23.9 U/mlMPMS (1-methoxy-5-methyl-phenazinium methyl sulfate). This isfreeze-dried and reconstituted in 13 ml of 144 μm NBT (nitrobluetetrazolium) in 1.15M Trizma, pH 7.6 G) Calibrator To make 1 ml ofcalibrator L-Homocysteine 0.86 mg 6M HCl 0.50 μl Milli-Ro water 9.50 μl

[0108] This is added to 990 μl of phosphate buffered saline (0.01mphosphate buffer containing 0.0027M KCL, 0.137M NaCl, pH 7.4). This isdiluted further with phosphate buffered saline to give stable liquidcalibrations of 2.5 μM, 5 μM, 10μM and 20 μM, L-homocysteine whichcorresponds to 5, 10, 20 and 40 μM L-homocysteine.

[0109] H) Stop Solution

[0110] 6M HCl.

EXAMPLE 2

[0111] Assay Protocol

[0112] Human blood was collected into vacutainer tubes containingcitrate. Plasma was separated from cells upon centrifugation at 1000× gfor 10 minutes at 2-8° C.

[0113] 10 μl of sample is mixed with 10 μl of 0.47% Hydrogen peroxide ona microtitre plate and incubated at room temperature for 3 minutes. 25μl of Enzyme Reagent 1 is added and incubated for 30 minutes at 37° C.10 μl of the same sample is mixed with 10 μl of 0.47% Hydrogen peroxideand incubated at room temperature for 3 minutes. 25 μl of Blank Reagent1 is added and incubated for 30 minutes at 37° C. Following thisincubation 85 μl of Reagent 2 is added to each and after mixing they areincubated a further 3 minutes at room temperature.

[0114] Reagent 2 contains the DTT binding agent and the acid destroysthe excess NADH. 125 μl of reagent 3 is added and incubated at 37° C.for 15 minutes. Reagent 3 brings the pH of the reaction to pH 7.0 andallows ADH to convert ethanol to acetaldehyde thus generating NADH. TheNADH is then converted to NAD+ as the colourless tetrazolium salt isconverted into the coloured product, aided by the oxidising agent, MPMS.The reaction is stopped by the addition of 15 μl of 6M HCL and thesample is read at 550 nm. The reading obtained for the sample treatedwith Blank Reagent 1 is subtracted from the reading for the sampletreated with Enzyme Reagent 1.

[0115] Calibrators are assayed by the same method and a calibrationcurve is constructed using the delta reading (the reading obtained forthe calibrator assayed in the presence of Blank Reagent 1 is subtractedthat obtained for the same calibrator assayed in the presence of EnzymeReagent 1) plotted against the known concentration of the calibrators.The Delta reading obtained for the sample is read from the standard lineand a concentration of homocysteine assigned.

EXAMPLE 3

[0116] Background Removal

[0117] The assay is performed as in Example 2, with the exception of thepre-treatment of samples with Hydrogen peroxide and the absence ofCatalase in Reagent 1 for one set of samples. Table 1 represents samplesassayed in the presence and absence of H₂O₂/Catalase. Each sample wasassayed four times with Enzyme reagent 1 and four times with BlankReagent 1. The concentration of homocysteine in each sample was obtainedby subtracting the mean reading obtained for each sample assayed withBlank Regent 1 from the individual readings obtained for 22each sampleassayed in the presence of Enzyme Reagent 1. The results presented intable 1 demonstrate that the background is reduced when samples wereassayed in the presence of hydrogen peroxide and Catalase. The reductionin background has improved the precision of the assay by decreasing thepercentage CV (coefficient of variance). TABLE 1 Sample Background andPrecision Obtained in the Absence of Hydrogen Peroxide/Catalase Presenceof Absence of H₂O₂/Catalyse H₂O₂/Catalyse Sam- Background [HCY]Background [HCY] ples reading μM % CV reading μM % CV 1 1.30 3.6 57.50.27 8.2 8.9 2 1.24 2.5 63.0 0.24 17.9 5.9 3 1.20 5.1 40.8 0.16 13.5 3.34 1.00 7.4 19.7 0.15 12.6 9.4 5 0.82 22.5 11.4 0.22 38.1 1.4 6 1.15 3.964.6 0.17 10.8 4.3

[0118] The results presented in Table 1 were also assayed by the AbbottImx® Homocysteine assay. The Homocysteine concentrations obtained forthese samples are represented in Table 2 when H₂O₂/Catalase was notpresent the correlation was found to be R²=0.89 and in the presence ofH₂O₂/Catalase the correlation was R²=0.99. TABLE 2 Homocysteineconcentration of samples as determines by Abbot Imx Assay. SampleHomocysteine Concentration (μM) 1 8.3 2 15.3 3 13.8 4 11.4 5 39.1 6 11.2

EXAMPLE 4

[0119] Assay Performance in Comparison to Current Techniques

[0120] The assay was performed as in Example 2. A selection of plasmasamples including samples from patients with renal failure, samples fromhealthy volunteers from two sites collected in either citrated or EDTAvacutainers were assayed in the presence of H₂O₂/Catalase. Each samplewas assayed four times in the presence of Enzyme Reagent 1 and fourtimes in the presence of Blank Reagent 1. The concentration ofhomocysteine in each sample was obtained by subtracting the mean readingobtained for each sample assayed with Blank Reagent 1 from theindividual readings obtained for each sample assayed in the presence ofEnzyme Reagent 1. The concentrations obtained were correlated with thoseobtained in the Abbot Homocysteine IMX assay. The correlation was foundto be R²=0.96 and the results are represented in table 3. TABLE 3Homocysteine Concentrations. % CV of samples treated with H₂O₂ andCatalase Mean Specific values blank (in the (obtained in the presence ofpresence of Sample enzyme reagent 1) blank Estimated Imx No. + + + +reagent 1) conc. (μM) % CV value A22 0.291 0.29 0.293 0.284 0.21 10.84.7 7.63 A26 0.216 0.214 0.214 0.218 0.138 11 2.5 6.65 A29 0.231 0.2170.235 0.23 0.137 13.07 8.7 12 A31 0.282 0.28 0.284 0.276 0.168 15.9 2.710.97 A32 0.275 0.269 0.281 0.272 0.161 15.9 4.1 11.5 A36 0.279 0.2840.281 0.272 0.206 10 6.6 7.55 A39 0.298 0.208 0.304 0.296 0.193 15.3 4.68.67 A40 0.286 0.286 0.284 0.286 0.189 12.9 1 9.18 A41 0.255 0.263 0.2440.265 0.154 14.67 9.4 A42 0.229 0.23 0.222 0.222 0.154 10.16 6.2 6.52A44 0.247 0.248 0.243 0.243 0.139 13.4 2.6 7.57 A45 0.249 0.254 0.2510.253 0.173 9.8 3.1 10.9 B47 0.411 0.411 0.423 0.404 0.277 17.9 5.718.82 B57 0.53 0.533 0.55 0.569 0.185 55.8 4.9 55.25 B73 0.365 0.3750.368 0.326 0.186 24.7 6.4 20.4 C8002762 0.516 0.527 0.521 0.527 0.26440.1 2 42.47 C8002524 0.396 0.413 0.375 0.396 0.221 27.1 8.8 26.48C8002384 0.429 0.423 0.429 0.426 0.249 27.6 1.6 27.13 C8002610 0.4260.444 0.445 0.438 0.236 26.8 4.2 27.69 C8002923 0.408 0.391 0.401 0.390.193 26.9 4.1 25.83 C8003302 0.257 0.258 0.249 0.253 0.171 11.2 4.8110.18 C8003316 0.33 0.334 0.353 0.334 0.247 15.1 8.9 12.98 C80032000.358 0.356 0.355 0.36 0.174 25.8 1.3 2156 C8002545 0.448 0.461 0.4660.472 0.226 33.5 4.5 32.78 C8002369 0.499 0.49 0.498 0.475 0.268 31.65.2 30.1 C375262 0.29 0.301 0.307 0.307 0.176 17.88 6.5 15 C485969 0.2190.229 0.216 0.228 0.145 11.13 8.4 9.48 C357556 0.355 0.338 0.347 0.3290.221 17.3 9.4 17.4 C515036 0.358 0.341 0.358 0.363 0.234 16.7 7.2 15.61C513311 0.303 0.303 0.288 0.285 0.164 18.2 6.7 13.81 C483375 0.341 0.3510.355 0.366 0.185 22.9 5.7 23.52 C489490 0.24 0.231 0.237 0.24 0.12415.9 3.4 13.34 D190599 25 0.25 0.256 0.255 0.244 0.149 13 5.8 D190599 320.168 0.17 0.169 0.175 0.11 7.4 5.4 9.48 D190599 28 0.217 0.21 0.2140.212 0.151 7.9 5.6 D190599 29 0.276 0.28 0.282 0.281 0.147 18.4 1.8D190599 30 0.202 0.2 0.2 0.195 0.134 8.2 5.3 D190599 31 0.197 0.2020.196 0.194 0.126 11.2 4.6 7.4 E701003 0.248 0.253 0.241 0.243 0.167 9.97.5 E701008 0.359 0.361 0.366 0.359 0.231 17 2.7 11.72 E701005 0.2540.242 0.254 0.255 0.178 9.4 9.7 6.25 E701006 0.302 0.306 0.301 0.2930.189 15 5.4 13.04 E701004 0.225 0.231 0.23 0.228 0.146 12.9 3.1 9.06

[0121] The correlation between obtained values and values obtained viaImx have been plotted on FIG. 2.

EXAMPLE 5

[0122] Comparative Assay Performance in the Presence/Absence of BSA andDTT

[0123] Background signals for homocysteine assays were determined for acentrifuged but unfiltered plasma sample using the LDH andtetrazolium/formazan system of Examples 1 and 2 under three differentassay conditions: (A) the blank enzyme reagent 1 contained BSA as thecryo/lyoprotectant, and glutamate, alanine aminotransferase,pyridoxyl-5-phosphate and maleimide were not used; (B) the blank enzymereagent 1 contained BSA as the cryo/lyoprotectant, glutamate, alanineaminotransferase and pyridoxyl-5-phosphate were not used; and (C) theblank enzyme reagent 1 was used in non-lyophilized solution form with noBSA present, and reagent glutamate, alanine aminotransferase andpyridoxyl-5-phosphate were not used.

[0124] The background signals for incubation times of 15 to 40 minutesfor (A) ♦, (B) ▪ and (C) ▴ are shown in FIG. 1 of the accompanyingdrawings.

EXAMPLE 6

[0125] Assay Protocol

[0126] The assay was performed as in Example 2, however in place ofpre-treatment of sample with Hydrogen peroxide and the inclusion ofCatalase in Reagent 1, the sample is pre-treated with PyruvateCarboxylase.

[0127] The sample is mixed with the following combination of reagents:100 mM trizma base, pH 7.5, 1 mM ATP (adenosine tri-phosphate), 5 mMmagnesium chloride, 15 mM sodium or potassium bicarbonate, 0.1 mM acetylcoenzyme A, 0.117 U/ml pyruvate carboxylase and incubated for 90 minutesat 37° C.

[0128] The results from one such study are depicted graphically on FIG.3. The signals for varying homocysteine concentration of 0 to 100 μM areshown.

EXAMPLE 7

[0129] Assay Protocol

[0130] The assay is performed as in Example 2, however in place ofpre-treatment of sample with hydrogen peroxide and the inclusion ofcatalase in Reagent 1, the plasma or serum sample is filtered through a30 kD exclusion filter and centrifuged at 10,000× g for 10 minutes. Theassay then proceeds as before. The results showing the reduction inbackground signal are represented on table 4. TABLE 4 background signallevels before and after filtration. Background Background reading beforereading after Sample filtration filtration 7 0.18 0.13 8 0.21 0.14

EXAMPLE 8

[0131] Assay Protocol

[0132] The assay is performed as in Example 2, however in place ofpre-treatment of sample with hydrogen peroxide and the inclusion ofcatalase in Reagent 1, the sample is pre-treated with pyruvate oxidase.

[0133] The sample is mixed in a 1:1 ratio with pyruvate oxidase at 15U/ml, 5 mM magnesium chloride, 0.1 mM thiamine pyrophosphate, 0.1 mMflavin adenine dinucleotide and incubated at 37° C. for 30 minutes.

EXAMPLE 9

[0134] Removal of Background

[0135] The assay is performed as in Example 2, however in addition topre-treatment of the sample, especially a serum sample, the sample isheat treated at 40-60° C. for 15 to 60 minutes. The assay proceeds asbefore following this step. This additional step removes some of thebackground left following the removal of Pyruvate and keto acids. Theresults are represented on table 5. TABLE 5 background signal levelsbefore and after heat treatment. Background Background reading beforereading after Sample heat treatment heat treatment 9 0.51 0.26 10 0.340.23 11 0.35 0.26

EXAMPLE 10

[0136] Passive Coating Method

[0137] HDS mixed with 0.1M phosphate buffer containing 0.1M NaCl. 50 μlto 300 μl of this solution is added to a microtitre plate, and isincubated for 2 hours. The microtitre plate is then washed 3 times withthe same buffer solution and dried in a tunnel drier. To improvestability, an overcoat is added and consists of phosphate buffer (0.1Mto 0.5M) containing sucrose (1-10%).

[0138] Assay Using Immobilized HDS

[0139] 1 mM DTT is added to the sample, calibrator or control in orderto cleave homocysteine from binding partners. The treated sample is thenadded to the plate, and incubated for 3 minutes. The plate is thenwashed with phosphate buffer.

[0140] The assay is performed as in Example 2, however, Reagent 1 doesnot contain HDS as it is bound to the plate.

EXAMPLE 11

[0141] Assay Protocol

[0142] The assay is performed as in Example 2, however in place ofpre-treatment of sample with hydrogen peroxide and the inclusion ofcatalase in Reagent 1, the plasma or serum sample is pretreated with20.8 μg/ml LDH and 1 mM NADH and incubated for 30 minutes at 37° C. TheNAD+ generated can be destroyed by treatment with Nitrous acid orconverted into NADH using 100 U/ml ADH and 5% ethanol.

[0143] The ADH can be immobilised on a Sepharose support before it ismixed with the sample and therefore removed from the plasma or serum bycentrifugation (2 min. at 2000× g). (Alternatively if ADH is notattached to a Sepharose support its excess activity can be removed by aninhibitor (e.g. tetramethylthiuram disulfide an inhibitor of yeastADH.))

[0144] Results obtained are represented on table 6, using immobilisedADH. The reduction in background signal is represented kinetically witha reduction in slope. TABLE 6 The effect of LDH pre-treatment and NAD +removal on background Slope (mAU/min Sample at 550 nm) In the presenceof 14 immobilised ADH In the presence of 88 immobilised ADH

1. An assay for homocysteine which comprises contacting a biologicalfluid sample with a reducing agent and subsequently with homocysteinedesulphurase, characterised in that said sample is contacted with anagent which binds, oxidizes or depotentiates said reducing agent afterbeing contacted with said homocysteine desulphurase.
 2. A homocysteineassay which comprises contacting a biological fluid sample with a liquidreagent containing a homocysteine converting enzyme, wherein saidreagent is produced by adding an aqueous liquid to a lyophilisatecontaining said enzyme and a cryo/lyoprotectant, characterised in thatsaid lyophilisate is substantially free of thiol-containingcryo/lyoprotectants.
 3. A homocysteine assay which comprises contactinga biological fluid sample with a liquid reagent containing homocysteinedesulphurase, wherein said liquid reagent is an aqueous liquidcontaining homocysteine desulphurase, a thiol-reducing reagent, and aproteinaceous or non-proteinaceous stabilizer.
 4. A homocysteine assaywhich comprises contacting a biological fluid sample with a homocysteineconverting enzyme, characterised in that before contact with said enzymesaid sample is treated with an agent which serves to deactivatepyruvates, e.g. by immobilizing, binding or converting pyruvates.
 5. Ahomocysteine assay which comprises contacting a biological fluid samplewith an immobilized homocysteine converting enzyme, wherein saidbiological fluid sample contacts the immobilized enzyme under such timeand conditions to allow the homocysteine in the sample to bind to saidenzyme, characterised in that the biological fluid sample is thenremoved from the assay.
 6. A homocysteine assay which comprisescontacting a biological fluid sample with a homocysteine convertingenzyme, characterised in that before contact with said enzyme saidsample is filtered through an exclusion filter and centrifuged in orderto remove pyruvates.
 7. An assay as claimed in at least two of claims 1to
 6. 8. An assay as claimed in at least three of claims 1 to
 6. 9. Anassay as claimed in at least four of claims 1 to
 6. 10. An assay asclaimed in claims 1, 2 and
 4. 11. An assay as claimed in claims 1, 3 and4.
 12. An assay as claimed in claim 4 wherein the agent which serves todeactivate pyruvates is hydrogen peroxide.
 13. An assay as claimed inclaim 12 wherein the hydrogen peroxide is neutralised prior tocontacting the sample with said homocysteine converting enzyme usingcatalase.
 14. An assay as claimed in any one of claims 4, 12 or 13wherein after the sample is treated with the said agent, the sample isheated at 40-60° C. for 15 to 60 minutes prior to contacting with saidhomocysteine converting enzyme.
 15. An assay as claimed in claim 4wherein the agent which serves to deactivate pyruvates is pyruvatecarboxylase.
 16. An assay as claimed in claim 4 wherein the agent whichserves to deactivate pyruvates is pyruvate oxidase.
 17. An assay asclaimed in claim 4 wherein the agent which serves to deactivatepyruvates is lactate dehydrogenase.
 18. An assay as claimed in claim 6wherein the sample is filtered with a 30 kD exclusion filter.
 19. Anassay as claimed in any one of claims 1 to 18 wherein said homocysteineconverting enzyme is HDS and wherein a NAD⁺/NADH cycling reaction isused to generate a coloured compound the concentration of which may becorrelated to the concentration of homocysteine in the initialbiological fluid sample.
 20. A kit for a homocysteine assay, said kitcomprising: homocysteine desulphurase, preferably (i) in lyophilizedform, the lyophilisate being substantially free of thiol-containingcryo/lyoprotectants or (ii) in aqueous liquid form further containing adithiol reducing agent (e.g. DTT, DTE or TCEP) and a proteinaceous ornon-proteinaceous stabilizer; a homocyst(e)ine standard, preferably aplurality of standards containing HCy or homocystine at a plurality ofknown concentrations; a reducing agent, e.g. dithiothreitol,dithioerythiol, TCEP or methyl iodide; an agent which binds, oxidizes ordepotentiates the reducing agent, e.g. an organic disulphide or adithiol binding agent, preferably a maleimide; optionally one or morefurther reagents capable of converting the homocysteine conversionproduct of homocysteine desulphurase into a detectable analyte;preferably a pyruvate deactivating agent, e.g. hydrazine, acetoacetatedecarboxylase, pyruvate carboxylase, hydrogen peroxide or pyruvatedehydrogenase; optionally a filter for removing pyruvate, i.e. amolecular sieve; and optionally a filter capable of removing red bloodcells from blood.